Method of photometric in vitro determination of a blood gas parameter in a blood sample

ABSTRACT

A method of photometric in vitro determination of at least one blood gas parameter in a sample of whole blood. The whole blood sample is obtained by connecting an at least partially transparent sample container to an in vivo locality and transferring whole blood into the sample container, then breaking the connection. The sample container is arranged in an optical system which has a radiation source and a means for detecting radiation to locate the sample container between the radiation source and the radiation detection means. Radiation is transmitted to the sample from the radiation source and radiation emitted from the sample is transmitted to the radiation detection means. The detected radiation is used to determine the blood gas parameter of the sample. A system for use in this method has a radiation source, a radiation detection means, an at least partially transparent sample container, and a sample container station.

FIELD OF INVENTION

The present invention relates to a method of photometric in vitrodetermination of a blood gas parameter in a blood sample.

BACKGROUND OF THE INVENTION

Photometric analysis of the blood gas parameters pH, oxygen (O₂) andcarbon dioxide (CO₂) is in itself prior art, which is described indetail in a large number of publications. A representative selection ofthese publications is listed below.

Photometric determination of the oxygen concentration in blood or othermedia by the so-called luminescence quenching is thus known from i.e.,

Bacon, J. R. and Demas, J. N., "Determination of oxygen concentrationsby luminescence quenching of a polymer immobilized transition-metalcomplex", Anal. Chem., 59, 1987, 2780-2785,

Longmuir, I. S. and Knopp, J. A., "Measurement of tissue oxygen with afluorescent probe", Journal of applied physiology, 41, 1976, 598-602,

Waughan, W. M. and Weber, G., "Oxygen quenching of pyrenebutyric acidfluorescence in water. A dynamic probe of the microenvironment",Biochemistry, 9 (3), 1970, 464-473,

Bergman, I., Nature 218, 1958, 376,

Stevens in the specification of U.S. Pat. No. 3,612,866,

Stanley in the specification of U.S. Pat. No. 3,725,658,

Bacon, J. R. and Demas, J. N. in the specification of British patentapplication GB 2132348,

Peterson et al. in the specification of U.S. Pat. No. 4,476,870,

Buckles, R. G. in the specification of U.S. Pat. No. 4,399,099,

Hirschfeld, T. in the specification of U.S. Pat. No. 4,542,987,

Dukes et al., in the specification of U.S. Pat. No. 4,716,363,

Lubbers et al. in the specification of U.S. Pat. No. Re. 31,879,

Kahil et al. in the specification of International patent application WO87/0023,

Murray, R. C., Jr. and Lefkowitz, S. M. in the specification of Europeanpatent application EP 190829,

Murray, R. C., Jr. and Lefkowitz, S. M. in the specification of Europeanpatent application EP 190830, and

Hesse, H. C. in the specification of East German patent DD 106086.

Determination of the carbon dioxide content in blood by irradiating with4,26 μm radiation is known from:

Manuccia et al. in the specification of U.S. Pat. No. 4,509,522,

Mosse, C. A. and Hillson, P. J. in the specification of British patentapplication GB 2160646, and

Nestor, J. R. in the specification of European patent application EP253559.

Determination of pH in blood by contact with a pH indicator is knownfrom, i.e., the following publications:

Seitz, W. R. and Zhujun, Z. in the specification of U.S. Pat. No.4,548,907,

Wolfbeis, O. S. et al., "Fluorimetric analysis. 1. A study offluorescent indicators for measuring near neutral ("physiological")pH-values", Fresenius Z. Anal. Chem. 1983, 314, 119-124,

Peterson, J. I. et al., "Fiber optic pH probe for physiological use",Anal. Chem. 1980, 52, 864-869,

Kirkbright, G. F. et al., "Fiber optic pH probe based on the use of animmobilized colorimetric indicator", Analyst 109, 1984, 1025-1026, and

Gerich I. L. et al., "Optical fluorescence and its application to anintravascular blood gas monitoring system", IEEE Transactions onBiomedical Engineering 2, 1986, 117-132.

Determination of the intraarterial values of all three blood gasparameters by means of a fluorescence based measuring system is knownfrom Miller et al,. "Performance of an in-vivo, continuous blood-gasmonitor with disposable probe", Clin. Chem. 33 (9), 1987, 1538-1542.Extracorporeal determination of all three parameters by means of an alsofluorescence based measuring system Gas-STAT™, produced byCardiovascular Devices Inc., USA, is finally described in brochuresconcerning this system and in the article by Clark, C. L., "Earlyclinical experience with Gas-STAT", J. Extracorporeal Technol., 18 (3),1986, 185-189. The determination of the blood gas parameters proceedscontinuously in the Gas-STAT™ system. Inside a cuvette, which isinserted in the extracorporeal circulation established at a cardiacoperation, fluorescence based sensors are placed. Via optical fibersexcitation radiation is provided and emitted fluorescence radiation istaken away. The intensity of the latter depends of the concentration ofthe matter measured by the relevant sensor.

None of these publications relating to photometric analysis of the bloodgas parameters describes an in vitro method for determination of one orseveral blood gas parameters in discrete samples and based on simplesample handling principles.

However, in vitro determination of the blood gas parameters pH, oxygen,and carbon dioxide in a blood sample has so far mostly been performed bymeans of blood gas analyzers as, e.g. the blood gas analyzers producedand sold by Radiometer A/S, Copenhagen, under the name ABL Acid-BaseLaboratory.

These analyzers are mechanically complex, since the blood samples i.a.have to pass through the very fine fluid conduits of the analyzer, inwhich conduits electrochemical sensors are built-in. Blockage in theconduits or coatings on the active surfaces of the sensors can easilyoccur and interfere in or destroy a measurement.

On account of these circumstances the existing equipment requiresfrequent maintainance performed by specially trained personnel, and theequipment will normally be placed in a laboratory situated at a certaindistance from the patient. A period of reply of more than 10 min. andnormally up to half an hour from the time of the sampling to the momentof the analysis result being present is therefore not unusual. Beyondthat the waiting period can be unfortunate in connection with themedical treatment of the patient, the relatively long waiting periodalso has the consequence that the sample is to be kept cooled down toapp. 0° C. This is due to the fact that at higher temperatures themetabolic processes of the blood will cause changes in the blood gasparameters during the relevant periods.

Another disadvantage of the existing equipment is that there exists acertain risk for the operator to get in touch with sample residue withthe health risks this may imply in the form of transfer of infections,etc.

SUMMARY OF THE INVENTION

The object of the invention is to provide an in vitro method fordetermination of a blood gas parameter, the method being moreappropriate for the user in that there is obtained both a more simpleand less risky sample handling and a more simple maintenance of theanalyzer.

The method according to the invention is characterized in that the bloodsample is transferred directly from an in vivo locality to an at leastpartially transparent sample container, that the connection between thesample containing sample container and the blood circulation is broken,that subsequently the sample container with its content of blood sampleis brought into optical communication with an optical system adapted tothe relevant blood gas parameter and comprising a radiation source and aradiation detector interacting therewith, and that the blood gasparameter is determined on the basis of the radiation detected by theradiation detector.

In a preferred embodiment the method is characterized in, that theoptical communication is provided by placing the sample container in asample container station in an analyzer.

Alternatively, the optical communication can be established by one orseveral cables, which via contact elements to the sample container andoptical fibres establish optical communication between the opticalsystem and the sample container.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of the preferred embodiment of an analyzerand a sample container, which together constitute the system accordingto the invention for photometric in vitro determination of a blood gasparameter in a blood sample;

FIG. 2 is an enlarged schematic view from the above sample containerstation of the analyzer with the sample container;

FIG. 3, FIG. 4 and FIG. 5 are views of a preferred embodiment of asample container for the system according to the invention;

FIG. 6 is an electric block diagram of the analyzer shown in FIG. 1;

FIG. 7 shows the photometric basis for determination of the blood gasparameter pH;

FIG. 8 shows the photometric basis for determinatioin of the blood gasparameter CO₂ ;

FIG. 9 shows the photometric basis for determination of the blood gasparameter O₂ ;

FIG. 10 shows the photometric basis for determination of the hemoglobincontent;

FIG. 11 is a perspective view of a sample container for use indetermining pH;

FIG. 12 is a perspective view of a sample container for use indetermining CO₂ or hemoglobin;

FIG. 13 is a perspective view of a sample container for use indetermining O₂ ;

FIG. 14 is a perspective view of a holder for the sample container shownin FIG. 12;

FIG. 15 is a partial cross section of an optical unit in a systemaccording to the invention for photometric determination of pH and witha schematic representation of the components forming parts of theoptical unit;

FIG. 16 is a partial cross section of an optical unit in a systemaccording to the invention for photometric determination of CO₂ and witha schematic representation of the components forming parts of theoptical unit;

FIG. 17 is a partial cross section of an optical unit in a systemaccording to the invention for photometric determination of O₂ and witha schematic representation of the components forming parts of theoptical unit;

FIG. 18 is a partial cross section of an optical unit in a systemaccording to the invention for photometric determination of hemoglobinand with a schematic representation of the components forming parts ofthe optical unit;

FIG. 19 is a block diagram of the electronic circuit coupled to theoptical unit for photometric determination of O₂.

In the different figures like reference numerals designate like parts.

DETAILED DESCRIPTION OF THE INVENTION

The above mentioned less risky sample handling is i.a. a consequence ofthe possibility of removing the practically closed sample container withits content of blood sample after the termination of the analysisprocedure. This removal is a sanitarily appropriate arrangement,which--in relation to the methods, by which a sample is transferred fromthe sampling container to an analyzer and from there to a wastecontainer--reduces the risk for the user to get in touch with possiblyinfected sample residues.

The actual transfer of the blood sample from the sampling device to themeasuring apparatus according to the prior art is a not unessentialsource of error within blood gas analysis. This source of error iseliminated by the method according to the present invention.

Apart from the more simple and less risky sample handling and thereduced maintenance of the analyzer obtained by the method according tothe present invention, there is also in other ways obtained a simplifiedanalysis procedure compared to the current blood gas analysismethodology.

The current methodology using equipment based on electrochemical sensorsnormally involves a relatively frequent calibration of the sensors. Bytraditional blood gas analyzers calibration routines are prescribed,whereby the sensors with an interval of 1 to 2 hours are contacted by aliquid or gaseous calibration medium with a specific content of therelevant parameters. The calibration medium is discarded after use andthe operator therefore has to secure the presence of the necessarycalibration medium. By the realization of the method according to theinvention this calibration medium consuming calibration process can beavoided.

Locally, the sample container has to communicate optically with theradiation source and the radiation detector, both of which preferablyare located outside the sample container, and it therefore has to bemade of a material which is transparent for the relevant radiation atleast in the areas communicating with the radiation source and theradiation detector. The material also has to provide the samplecontainer with a sufficient diffusion tightness for oxygen and carbondioxide, which means that the content of oxygen and/or carbon dioxidemay not change substantially during the time normally passing from thesampling to the moment of the analysis. A polymeric base material, ifnecessary with a coating of a polymeric or metallic gas barrier sheet issupposed to be suitable and the base material is preferably an injectionmouldable material. A suitable base material is polyethyleneterephtalate (Arnite™ from AKZO, Arnhem, Holland).

As to the handling of samples for clinical chemical analysis anequipment with a cavity sufficiently small for a given sample to besucked into the cavity by capillary effect is known from thespecification of International patent application WO 86/00138 (Shanksel. al.).

In this equipment the cavity is provided with an electrode structure andpossibly a coating of a material adapted to the analysis to be performedwith the equipment. The electrode structure provided in the cavity maybe a potentiometric ion sensitive electrode structure or an amperometricelectrode structure. The latter is described in connection withdetermination of hydrogen peroxide and oxygen in the sample.Supplementary use of the equipment for optical analysis of the productsof a specific binding reaction is also described.

From the specification of Danish patent publication no. 150804 (Lilja,J. E. and Nilsson, S. E. L.) is known a sample container for sampling,mixing a sample with at least one reagent and directly performing aseparate optical analysis of the sample mixed with the reagent. Thesample container has a capillary cavity coated with a reagent and theinlet to the sample container works by capillary effect. The samplecontainer is stated to be useful for most different kinds of analysisand to be especially advantageous for determination of hemoglobin.

From the specification of British patent application GB 2 025 065(Meiattini, F. et. al.) is known a plunger syringe for withdrawal of ablood sample. The blood sample is analysed by means of sensorsincorporated in the syringe plunger. It is thereby avoided to transferthe sample to a sample station.

The sensors are adapted for connection with an analyzer via conductorsfor registering, processing, and outprinting analysis data. The specificsensors described in the specification of the said British patentapplication GB 2 025 065 are electrochemical sensors for blood gases andblood electrolytes.

Since the method according to the invention is based on photometricprinciples, the connection of electric conductors to the sensors of thesample container, which is nescessary when the sensors areelectrochemical sensors as in the above described sample containers, isavoided. A more simple design of the sample container and of theinterface between the sample container and the respective analyzer isthereby possible.

It shall finally be mentioned that the technological basis alsocomprises other clinical chemistry analyzers consisting of a combinationof disposable components, which are only used for one single analysisoperation and only get in touch with one single sample, and an analysingsection adapted for receiving the sample containing disposable deviceand containing the additional components necessary for accomplishing aclinical chemical analysis. Special blood gas analyzers are, however,not known among these.

"Photometric determination" denotes in the present context everydetermination based on measuring changes in electromagnetic radiation,which under controlled conditions is emitted, transmitted, absorbed, orreflected.

"In vivo locality" denotes in the present context a locality being indirect connection with the blood circulation or being a locality in theblood circulation itself. Sampling by arterial puncture, whereby theblood sample is transferred from the artery to the sample container by athin needle, as well as via an arterial catheter or via capillarypuncture are sampling methodologies, in which the blood sample istransferred directly from an in vivo locality to a sample container.

In the case where the blood sample is provided by capillary puncture,the use of a sample container with a dimension sufficiently small forthe sample container to be filled by capillary effect is preferred.

In the case where a sample of arterial blood is desired, the use of asample container with an inlet located in a coupling means, preferably aLuer cone, for coupling the sample container to a needle or a catheteris preferred.

In the case where the sampling of the blood sample is performed by useof a needle coupled to the sample container, it is especiallyadvantageous to provide the sample container with a needle protectingmeans integral therewith, preferably a jacket movable in the axialdirection of the sample container between a first position, wherein thejacket exposes the point of the needle, and a second position, whereinthe jacket surrounds the point of the needle.

With this preferred embodiment of the sample container it is possible toobtain sufficient security for the user against being injured by theneedle just by displacing the jacket into the second position. The useris thus not in risk of getting in touch either with the needle carryingpart itself or with the immediately surrounding area during removal orapplication of a protecting means.

Alternatively the needle protecting means can be an elongatedgully-shaped element pivotally mounted around an axis located near theinlet of the sample container. During sampling the needle protectingmeans surround the sample container, while the needle is exposed.

By rotation 180° C. around the axis the needle protecting means arebrought to surround the needle, while the sample container is exposed.

The invention also relates to a system for photometric in vitrodetermination of a blood gas parameter in a blood sample, the systemcomprising an at least partially transparent sample container andfurther comprising an analyzer with a sample container station and withan optical system adapted to the relevant blood gas parameter andcomprising a radiation source and a radiation detector interactingtherewith, the sample container station being so arranged in relation tothe radiation source and the radiation detector that a sample containercontaining blood sample and placed in the sample container station is inlocally optical communication with the radiation source and theradiation detector and with means for registering the radiation detectedby the radiation detector.

In a preferred embodiment the analyzer comprises data processing meansfor processing the registered radiation data for deriving the relevantblood gas parameter from these. Alternatively the analyzer is adaptedfor connection to a separate data processing unit.

In a further preferred embodiment of the system according to theinvention the analyzer comprises means for displaying the relevant bloodgas parameter or any possible parameters derived from this.Alternatively, the analyzer is adapted for connection with means suchas, e.g. a data screen, a display, a printer, or a plotter.

The invention will now be explained in the following with reference tothe drawings and the subsequent examples. In the drawings

The analysis system shown in FIG. 1 and generally designated 10 is acompact portable "stand-alone" system, which is suited for decentralizeduse, i.e. use outside a regular laboratory environment, e.g. in anoperating room or at an intensive ward. The analysis system 10 comprisesa blood sample container 2 for disposable use and used in connectionwith an analyzer 11. The sample container 2 is more explicitly describedin connection with the description of FIGS. 3-5 below. The samplecontainer 2 and the analyzer 11 are adapted to interact in the way thatthe analyzer 11 has a sample container station 1 with an optical section3 adapted for receiving the sample container 2, so that the opticalcommunication between the sample container 2 and the optical componentsof the optical unit 3, which is necessary for photometric analysis, isobtained.

The sample container station 1 can be closed by a cover 8, which isclosed after placing the sample container 2 in the station. By closingthe cover 8 different mechanisms are activated, e.g. a not shownclamping mechanism, which secures the sample container 2 in the opticalsection 3 and at the same time thermostatically controls the samplecontainer to a desired temperature, preferably app. 37° C.

Closing the cover 8 further results in a signal being sent to thecontrolling unit of the analyzer and indicating the start of an analysisprocedure. An operator can control the operation of the analyzer bymeans of a keyboard 5 and a display 6. The analyzer 11 preferably alsocomprises a printer, which can produce an outprint 7 of the analysisresults obtained by the analyzer.

After placing the sample container 2 in the sample container station 1and closing the cover 8 of this, the optical components comprising ofradiation sources and radiation detectors are activated, whereupon theanalyzer 11 calculates one or several blood gas parameters on the basisof the signals from the radiation detectors. The result of thecalculations is displayed on the display 6 and is printed on the paperoutprint 7 by the printer. When the calculations are terminated and theresults displayed and/or printed out the cover 8 is opened and thesample container 2 is displaced from the sample container station 1 andremoved.

In a larger scale FIG. 2 shows a partially schematic section of thesample container station 1 viewed from above. As shown the opticalsection 3 comprises four optical units 30, 40, 50, and 60 each adaptedfor determination of its relevant blood parameter. The sample container2 is placed in a slot 4 in the optical section 3.

The optical unit 30 contains the optical components necessary forphotometric determination of pH. The optical unit 40 contains theoptical components necessary for photometric determination of CO₂. Theoptical unit 50 contains the optical components necessary forphotometric determination of O₂ and finally the optical unit 60 containsthe optical components necessary for photometric determination ofhemoglobin. Even though the analyzer 11 is shown containing four opticalunits it can in principle contain an arbitrary number and/or anarbitrary combination of units, including units adapted fordetermination of other parameters than those mentioned here.

FIG. 3 shows a longitudinal section of the sample container 2 and foursegments of this in a larger scale. The sample container comprises abody 23, which at least in specified areas is made of a materialtransparent for the relevant radiation. The body 23 has a continuousconduit 22 locally extended for forming measuring chambers 300, 400,500, and 600. During a course of measurement the actual blood samplefills the conduit 22 from its inlet aperture 21 to a hydrophobic filter24 placed behind the measuring chambers. The section of the body 23surrounding the inlet aperture is provided with a Luer cone and istherefore suitable for being mounted with a needle 20 of the typenormally used for blood sampling. The section 25 of the body 23 pointingaway from the inlet aperture is adapted for coupling with a traditionalplunger syringe. Such a plunger syringe is used as an aid at thesampling in certain situations, e.g. when the patient, whose blood gasparameters are to be determined, has a very low blood pressure.

When the sample container 2 is placed correctly in the sample containerstation 1, the measuring chambers 300, 400, 500, and 600 communicateoptically with the optical units 30, 40, 50, and 60. The measuringchamber 300 optically communicating with the optical unit 30 is adaptedfor determination of pH in the blood sample and contains a cellophanemembrane 316, to which is immobilized a pH absorbance indicator. Whenthe indicator is in chemical equilibrium with the blood sample, therelation between the acid form of the indicator and the basic form ofthe indicator reflects the pH-value of the blood sample. The chemicaland photometric basis for the pH determination appears from FIGS. 7, 11,and 15 and of the description of these. An embodiment of the opticalunit 30 appears from FIG. 15 and the description of this.

The measuring chamber 400 communicates optically with is the opticalunit 40 adapted for determination of the carbon dioxide content in theblood sample. As it is seen from FIGS. 8, 12, and 16 and the descriptionof these, this determination takes place on the basis of thetransmission properties of the sample for radiation at the wavelength4260 nm. An embodiment of the optical unit 40 appears from FIG. 16 andthe description of this.

The measuring chamber 500 is the measuring chamber wherein thedetermination of the oxygen content of the sample takes place and thismeasuring chamber communicates optically with the optical unit 50. Onone of its surfaces the measuring chamber 500 has a PVC membrane 517dyed with the phosphorescent compound PdTFPP (palladium(II)-tetra(pentafluorphenyl)-porphyrin). The phosphorescent compound isexcited with radiation of a wavelength at app. 556 nm, and the oxygencontent is determined by determining characteristics of radiation at thewavelength 673 nm emitted from the excited phosphorescent compound. Thechemical and photometric basis for the oxygen determination appears fromFIGS. 9, 13, and 17 and the description of these. An embodiment of theoptical unit 50 appears from FIG. 17 and the description of this.

The measuring chamber 600 is the measuring chamber wherein thehemoglobin content and the oxygen saturation of the blood sample isdetermined. The measuring chamber is adapted to optically communicatewith the optical unit 60 and has a coating 610 of a chemical hemolysisagent on its internal surface. The hemoglobin content in the bloodsample is determined by determining characteristics of radiation at thewavelengths 506 nm and 600 nm transmitted through the blood sample. Thechemical and photometric basis for the determination of hemoglobinappears from FIGS. 10 and 18 and from the description of these. Anembodiment of the optical unit 60 appears from FIG. 18 and thedescription of this.

The exterior walls of the measuring chambers 300, 400, and/or 500 arepreferably made by a material different from the base material of thesample container 2. An ethylene vinylalcohol copolymer of the typeEVAL-E™ from Kuraray Co., Osaka, Japan is suited for application asmeasuring chamber walls due to its low oxygen and carbon dioxidepermeability and its adequate optical properties. Another suitablematerial for wall elements constituting the exterior walls of themeasuring chambers 300, 400, and/or 500 is glass.

Finally it appears from FIG. 3 that the sample container 2 has anintegral needle protecting means 26. In the embodiment shown the needleprotecting means 26 is a tubular jacket movable in the axial directionof the sample container between a first position, wherein the jacketexposes the point of the needle, and a second position, wherein thejacket surrounds the point of the needle.

FIG. 3 shows the protecting jacket 26 in the first position, in which itis placed at the sampling moment.

FIG. 4 shows a longitudinal section of the sample container. The sectionis placed perpendicular to the section shown in FIG. 3.

The sample container body 23 consists of two halves, of which only oneis visible in FIG. 4.

FIG. 5 shows finally the same section as FIG. 3 but in FIG. 5 theprotecting jacket is displaced to the second position, wherein itsurrounds the point of the needle. The jacket 26 is displaced to thissecond position immediately after the sampling.

FIG. 6 shows the electrical block diagram for the analyzer 11 and speaksfor itself.

FIG. 7 shows the absorption spectrum for a pH absorption indicator N-9more closely described below in connection with FIG. 11. The indicatoris immobilized on a cellophane membrane by the method also described indetail in connection with FIG. 11. The spectrum is recorded by aspectrophotometer of the type Shimadzu Spectrophotometer UV 250. Theabsorption measurements were performed on a cellophane membrane(6×12×0.028 mm) with immobilized indicator. The cellophane was placed inthe cuvette of the spectrophotometer in a holder adapted thereto andwith dimensions adapted to the dimensions of the cuvette.

In order to determine the absorption conditions at different pH-values,a number of pH buffers with pH values in the pH range from pH 6.14-11.74was produced. Each buffer consisted of 500 ml 0.1M KH₂ PO₄ to which wasadded the necessary amount of 0.1M NaOH. The pH value of each buffer waspotentiometrically measured (Radiometer PHM80) by means of a glasselectrode (Radiometer GK2402C).

It appears from the spectrum shown in FIG. 7 that the acid form of theindicator has an absorption top at 458 nm and that the basic form of theindicator has an absorption top at 595 nm and that the indicator notessentially absorbs radiation at wavelengths above 750 nm.

FIG. 8 shows an absorption spectrum for whole blood (hemoglobin content9 mmol/l, Pco₂ 419 mmHg) recorded on an IR spectrophotometer of the typeBeckman IR9. From the absorption spectrum it appears that a content ofCO₂ in the blood sample results in an absorption at about 4260 nm.

For each of three different oxygen levels in gaseous samples FIG. 9shows a signal representing the time dependent emission radiation from aluminophor excited with a modulated excitation source. It appears fromthe figure that the frequency of the signal depends on the oxygencontent. The figure is provided by use of the sample container accordingto FIG. 13 and the optical unit according to FIG. 17 with electronics asshown in FIG. 19 and an oscilloscope coupled thereto.

FIG. 10 shows the absorption spectra for oxyhemoglobin anddeoxyhemoglobin, respectively. The two absorption spectra intersect inan isobestic point situated at the wavelength 506 nm. The totalhemoglobin content of a blood sample can be determined on the basis ofthe transmission properties of the sample for radiation at thewavelength 506 nm, as Hb_(tot) is the sum of the content ofoxyhemoglobin and the content of deoxyhemoglobin. On the basis of thetransmission properties of the sample for radiation at anotherwavelength the content of, e.g., oxyhemoglobin can be determined andthereby also the oxygen saturation (oxyhemoglobin/Hb_(tot)). Theabsorption spectrum shown here is reproduced from Zijlstra, W. G. et al."Problems in the spectrophotometric determination of HbO₂ and HbCO infetal blood", Physiology and Methodology of Blood Gas and pH, volume 4,1984, 45-55.

FIG. 11 shows an embodiment of a sample container for use indetermination of pH in a blood sample. The sample container generallydesignated 3000, is intended to interact with an analyzer with anoptical unit as the one more closely described below in connection withFIG. 15. The sample container consists of two halves 3001 and 3002.These halves are made from a transparent plastic material, e.g.,softened polymethyl methacrylate of the type DEGALAN™ SZ70 (Superfos,Copenhagen, Denmark). The two halves are assembled by pins 3005 and 3006in the half 3001 engaging corresponding, not shown recesses in the half3002, while not shown pins in this engage recesses 3007 and 3008 in thehalf 3001. The two halves are hereafter welded together by ultrasonicwelding. The line 3009 of material outlined on the lower half 3001 shownin the figure forms a welding seam after the welding. This line ofmaterial lies along the edge of a longitudinal conduit 3004, whichcentrally expands transversely and forms a measuring chamber 3011. Awall section 3010 of the upper half 3002 shown in the figure has to beelastically deformable and for that reason has a very reduced wallthickness compared to the rest of the half. As mentioned in connectionwith FIG. 7 a cellophane membrane 3003, to which there is immobilized apH indicator, is placed in the measuring chamber 3011.

Preparation of the Cellophane Membrane with Immobilized pH Indicator

The pH indicator is delivered by Merck, Darmstadt, West Germany underthe name N-9 and is known to contain the reactive group

    --SO.sub.2 --CH.sub.2 --CH.sub.2 --O--SO.sub.3 H

The immobilization of the indicator to the membrane takes place in aprocess, whereby the above mentioned reactive group is transformed withsodium hydroxide into a vinyl sulphone group, which can be coupled tocellophane previously activated by conditioning the cellophane in 0.1MNaOH for 15 minutes.

The reaction scheme describing the coupling reaction between theindicator and the cellophane membrane is:

    R--SO.sub.2 --CH.sub.2 --CH.sub.2 --O--SO.sub.3 H+2 NaOH

    R--SO.sub.2 --CH═CH.sub.2 +Na.sub.2 SO.sub.4 +2 H.sub.2 O (1)

    R--SO.sub.2 --CH═CH.sub.2 +HO-cellophane

    R--SO.sub.2 --CH.sub.2 --CH.sub.2 --O-cellophane           (2)

R here designates an aryl group.

The quantitative immobilization procedure is the following: 100 mgcellophane (of the type 113650-36/32 from Struers, Copenhagen, Denmark)is after the above mentioned conditioning with NaOH prepared with anindicator solution consisting of 6 mg N-9, 1000 mg NaCl, 500 mg Na₂ CO₃,200 μl 8M NaOH in 40 ml H₂ O. The cellophane is left in the indicatorsolution for 30 minutes. Hereafter the cellophane is removed from theindicator solution and washed several times with deionized water,whereafter it is left in deionized water at least through the nightbefore use. The prepared membrane is then kept in deionized water untiluse.

FIG. 12 shows an embodiment of a sample container for use indetermination of the content of carbon dioxide in a blood sample. Thesample container, generally designated 4000, is intended to interactwith an analyzer with an optical unit as the one described below inconnection with FIG. 16. The sample container 4000 consists of twohalves designated 4001 and 4002, respectively. The two halves areassembled in the same way as the sample container 3000 according to FIG.11. After the assembly, the sample container has analogously hereto aninternal sample conduit 4006, which centrally expands transversely forforming a measuring chamber 4007. The two halves 4001 and 4002 are madefrom a plastic material, but the measuring chamber itself is in adirection perpendicular to the conduit 4006 defined by two glass platesor alternatively EVAL-E™ plates 4003 and 4004 secured between the twohalves. One or several very thin lines 4005 of material ensure a welldefined distance, e.g. 35 μm, between the two plates 4003 and 4004.

FIG. 13 shows a sample container for use in determination of the oxygencontent in a blood sample. The sample container, generally designated5000, is intended to interact with an analyzer with an optical unit asthe one described below in connection with FIG. 17. The sample container5000 consists of two halves designated 5001 and 5002, respectively. Thetwo halves are assembled in the same way as the sample container 3000according to FIG. 11. Thus, after the assembly the sample container hasa sample conduit 5006, which centrally expands transversely into ameasuring chamber 5007. The two halves 5001 and 5002 are made from aplastic material. One of the walls in the measuring chamber 5007consists of a glass plate 5003 in the form of a microscope cover glass,or alternatively an EVAL-E™ plate, on which there is cast a 2 μm coating5004 of PVC containing PdTFPP. The preparation of the element consistingof the plate 5003 and the PVC coating 5004 is more closely describedbelow. A double adhesive ring 5005 secures the plate 5003 to the samplecontainer part 5002.

Preparation of the Wall Element with Luminophor

A solution consisting of 15 mg PdTFPP (synthesized for the applicant forthe purpose), 199.5 mg PVC (BREON S 110/10; BP Kemi, Copenhagen,Denmark) and 1.5 ml tetrahydrofuran (LiChrosolv™; Merck, Darmstadt, WestGermany) is cast on a rotating microscope cover glass etched byhydrofluoric acid in a dry atmosphere by putting on 10 μl solution asdrops. The speed of rotation is 110-120 rotations/sec.

Less than two hours after the casting the wall element is placed at 90°C. in an incubator for 40 minutes, whereby the PdTFPP containing PVCcoating is hardened.

The membrane thickness is reproducible and is about 2 μm.

FIG. 14 shows a holder 7000 for the sample container 4000. The holderhas a recess 7001 adapted to the sample container 4000 and intended forreceiving this. The holder 7000 is intended to be placed in an opticalunit, e.g. the optical unit 40 according to FIG. 16 described below orthe optical unit 60 according to FIG. 18 described below. When theholder 7000 is placed in one of the optical units mentioned, a hole 7002secures the optical communication between the sample container and theradiation source. The holder 7000 is preferably made from a well heatconducting material, e.g. aluminium.

FIG. 15 shows a prototype of an optical unit 30 for use in determinationof pH in a blood sample. The blood sample is located in the measuringchamber 3011 in the sample container 3000, which is secured between astationary part 305 and a moving part 306. The previously mentionedmembrane with immobilized pH-indicator is provided in the measuringchamber. Before the measuring, the blood is pressed away from themeasuring chamber by pressing the moving part 306 against the adjacentwall of the measuring chamber 3011. Pressing out the blood sample fromthe measuring chamber results in the transmission conditions inside themeasuring chamber not being influenced by the blood sample itself.

The optical unit 30 works in following way: A radiation source 301 inthe form of a halogen lamp with a builtin lens of the type LNS-SE6-560from Hamai Electric Lamp Co. TTD, Tokyo, Japan emits a parallel beam ofbroadbanded radiation. This radiation is transmitted to a heat absorbingfilter 302 of the type KG 5 from Schott, Mainz, West Germany. Thisfilter eliminates radiation from the infrared range. The radiation istransmitted from the filter to a depolarizer 303 and from there throughthe measuring chamber 3011. A silicon photodiode 304 of the type SFH 212from Siemens, Munich, West Germany, receives radiation reflected fromdifferent surfaces inside the optical unit and is coupled to theradiation source 301 and ensures constant radiation intensity from this.

After passage through the measuring chamber 3011, the radiation isfocused by a lens 307 (φ12 mm; f 12 mm; Thermooptik Arnold GmbH & Co.,Weilburg, West Germany) onto three silicon photodiodes 310, 313 and 315,which are all of the type SFH 212 and situated in the focal plane of thelens 307. From the lens 307 the radiation is transmitted to a dichroicmirror 308 reflecting radiation of wavelengths less than 560 mm andtransmitting radiation of longer wavelengths. The dichroic mirror isdelivered by Optisk Laboratorium, Technical University of Denmark,Lyngby, Denmark. The short-waved part of the radiation passes through aband-pass filter 309 (centre value 458 nm; half band width 5.1 nm;Ferroperm, Vedbaek, Denmark) and is transmitted from the band-passfilter 309 to a silicon photodiode 310 of the type SFH 212. The part ofthe radiation from the lens 307 transmitted through the dichroic mirror308 is transmitted to another dichroic mirror 311 reflecting radiationof wavelengths less than 690 nm and transmitting the more long-wavedradiation. The dichroic mirror is again delivered by OptiskLaboratorium, Technical University of Denmark, Lyngby, Denmark. Thereflected radiation is transmitted from the dichroic mirror 311 througha bandpass filter 312 (centre value 589 nm, half band width 14.8 nm;Ferroperm, Vedbaek, Denmark) and from there to a silicon photodiode 313of the type SFH 212. The radiation transmitted through the dichroicmirror 311 is transmitted from here to a bandpass filter 314 (centrevalue 750 nm; half band width 10 nm; Ferroperm, Vedbaek, Denmark) andfrom there to a silicon photodiode 315 of the type SFH 212. The siliconphotodiodes 310, 313 and 315 emit a current signal representing theintensity of 458 nm, 589 nm and 760 nm radiation, respectively. On thebasis of these radiation intensities the pH value of the sample iscalculated.

More precisely the pH value of the sample is calculated in the followingway:

With different samples in the form of blood sample as well as the belowmentioned two pH buffers in the sample container is for each wavelengthλ₁, λ₂, λ₃ determined the current I_(background),λ measured on thecorresponding photodiode for a sample container containing a cellophanemembrane without immobilized indicator (i.e. a clear cellophanemembrane) and sample. This current corresponds to I₀ (cf. Lambert Beer'sLaw). From the knowledge of the dynamic area of the amplifier for theindividual photodiode the current I_(background),λ can be related to theabsorbance A_(background),λ, as

    A.sub.background,λ =k·log I.sub.background,λ

where k is a constant.

In the measuring situation where the sample container contains acellophane membrane prepared with pH indicator, the currentI_(measurement),λ also relating to A_(measurement),λ is determined,whereupon the "true" absorbance at the wavelength λ is determined as

    A.sub.λ =A.sub.measurement,λ -A.sub.background,λ,

cf. A=log I-log I₀.

To correct further for drift in the optical system, unclear samplecontainers or other variations not directly relating to the pHdetermination, the absorbances A.sub.λ1 and A.sub.λ2 are corrected withA.sub.λ3, and the "new" absorbance values are designated A'.sub.λ1 andA'.sub.λ2.

The relation to Lambert Beer's Law hereafter appears as:

    A'.sub.λ1 =A.sub.λ1 -A.sub.λ3 =ε.sub.λ1,HIn ·c.sub.Hin ·1+ε.sub.λ1,In.spsb.- ·c.sub.In.spsb.- ·1

    A'.sub.λ2 =A.sub.λ2 -A.sub.λ3 =ε.sub.λ2,HIn ·c.sub.HIn ·1+ε.sub.λ2,In.spsb.- ·c.sub.In.spsb.- ·1                                               (3)

where HIn designates the acid form of the pH indicator, In⁻ designatesthe basic form of the indicator, c_(HIn) and c_(In).spsb.- in theconcentration of the acid and the basic form, respectively, of theindicator, ε the extinction coefficient for the wavelength and compounddefined by the suffix, and 1 the pathlength of the radiation through thesample,

which can be inverted to:

    c.sub.HIn =k.sub.11 A'.sub.λ1 +k.sub.12 A'.sub.λ2

    c.sub.In.spsb.- =k.sub.21 A'.sub.λ1 +k.sub.22 A'.sub.λ2 (4)

The constants k₁₁, k₁₂, k₂₁, and k₂₂ are determined by measuringA.sub.λ1, A.sub.λ2 and A.sub.λ3 with two pH buffers with known pH valuesas samples. For each of these pH buffers the proportion c_(In).spsb.-/C_(HIn) is determined according to the Henderson Hasselbalch equation:

    pH=pk.sub.a +log [c.sub.In.spsb.- /c.sub.HIn ]             (5)

If it is further supposed that c_(In).spsb.- +c_(HIn) =1, the two setsof connected values of c_(In).spsb.- and c_(HIn) can be calculated. Ifthe corrected absorbance values are inserted in the equation set (4),four equations with the four unknown quantities k₁₁, k₁₂, k₂₁, and k₂₂are obtained, and subsequently these four quantities can be calculated.

By measuring unknown samples it is hereafter possible to determinec_(HIn) and c_(In).spsb.- from the equation set (4) and subsequently pHfrom equation (5).

FIG. 16 shows a prototype of an optical unit 40 for use in determinationof carbon dioxide in a blood sample. The blood sample is located in themeasuring chamber 4007 in the sample container 4000 placed in a holder7000, which further is placed in a unit, generally designated 3. Theoptical unit 40 contains a radiation source 401 in the form of a thermicradiation unit, more definitely a CrNi filament heated to app. 1000° C.The radiation source is produced for the purpose by the applicant.Radiation from the radiation source 401 is transmitted to a concavemirror 402 of the type 4001021 (φ10 mm; f 5 mm; Thermooptik Arnold GmbH& Co., Weilburg, West Germany).

The concave mirror 402 depicts the filament 401 into a slit 403, and theradiation is transmitted from here to a concave mirror 404. The mutualorientation of the slit 403 and the concave mirror 404 is, that the slit403 is situated in the focal point of the concave mirror. From theconcave mirror 404 produced by the applicant for the present purposeradiation is transmitted to a grating 405 of the type OEM 300-40000-2525from Optometrics, Leeds, England. The grating is a 300 lines gratingoptimized to app. 4 μm.

The grating 405 is pivotally mounted and is continuously rotated betweentwo end positions. An optical position detector 406 reads the positionof the grating 405. The position of the grating 405 determines thewavelength of the radiation, which at a certain moment passes throughthe sample container. The actual wavelength is in the range from 4220 to4310 nm. From the grating 405 the radiation is transmitted back to theconcave mirror 404 from where the radiation is reflected, and theradiation relevant to the measurement, i.e., the radiation subsequentlypassing the sample container, is transmitted through a LWP filter 407(long wave pass filter) of the type LP 3500-F from Spektrogon, Taby,Sweden. This filter transmits radiation at wavelengths greater than 3500nm. From the filter 407 the radiation is transmitted through themeasuring chamber 4007 and from there to a pyroelectrical detector 408of the type KRX11 from Philips, Eindhoven, Holland.

The pyroelectrical detector emits a voltage signal proportional to theintensity of the radiation incident on the detector. Detector signalsrepresenting the intensity of 4210 nm radiation, 4260 nm radiation, and4310 nm radiation are registered, so that by means of the positiondetector 406 it is ascertained when the grating is situated in the firstend position (corresponding to transmitting 4210 nm radiation throughthe sample), the center position (corresponding to transmitting 4260 nmradiation through the sample) and the second end position (correspondingto transmitting 4310 nm radiation through the sample), respectively, andregistration of the detector signals shall take place at these moments.

The optical unit 40 described here is intended for determination of CO₂by the so-called baseline analysis method. By this method iscalculationwise found an absorbance of a sample not containing CO₂ atthe wavelength, at which the CO₂ absorption top is situated. Thebaseline analysis method is comprehended by viewing FIG. 8. From thecurve for whole blood with Pco₂ 419 mm Hg and from the construction linemarked with points it is seen that the transmittance I'/I₀ of a samplefree of CO₂ can be estimated by interpolation between the radiationintensities at two wavelengths situated close to and outside theabsorption top, in the current case 4210 nm and 4310 nm. Designating theactual transmittance I/I₀ the carbon dioxide concentration [CO₂ ] iscalculated from Lambert Beer's Law in the following way:

    log I.sub.0 /I-log I.sub.0 /I'=εCO.sub.2,4260 ·[CO.sub.2 ]·1

where εCO₂,4260 is the extinction coefficient for CO₂ at 4260 nm and 1is the pathlength of the radiation through the sample.

FIG. 17 shows a prototype of an optical unit 50 for use in determinationof oxygen in a blood sample. The blood sample is located in themeasuring chamber 5007 in the sample container 5000.

The sample container is placed in a section, generally designated 3.

From a radiation source 501 in the form of a modulated green light diodeof the type HBG 5566X from Stanley Electric Co. Ltd, Tokyo, Japanradiation is transmitted through a SWP filter 502 (short wave passfilter), which is specially produced for the applicant for the presentpurpose, and which eliminates radiation of wavelengths greater than 580nm. From the filter 502 the radiation is transmitted to a dichroicmirror 503 of the type BSP600 from Optisk Laboratorium, TechnicalUniversity of Denmark, Lyngby, Denmark. The dichroic mirror 503 reflectsradiation at wavelengths less than 600 nm and thereby reflects theradiation from the green light diode. The radiation is reflected fromthe dichroic mirror to a convex lens 504 (φ9.9 mm; f 7.3 mm; ThermooptikArnold GmbH & Co., Wellburg, West Germany).

The mutual orientation between the lens 504 and the sample container5000 is so that the sample container 5000 is situated in the focal planeof the lens. The radiation is focused on the sample container by thelens 504, where it excites a luminophor provided in the measuringchamber 5007. The excited luminophor interacting with the oxygen of theblood sample emits more longwaved radiation 505, which is transmittedfrom the sample container through the lens 504 and the dichroic mirror503 to an edge filter 506 of the type RG665 from Schott, Mainz, WestGermany. The filter transmits radiation of wavelengths greater than 665nm. The radiation transmitted through the filter 506 falls onto asilicon photodiode 507 of the type SFH 216 from Siemens, Munich, WestGermany, and the photodiode emits a time dependent electrical signalrepresenting the actual radiation intensity. In FIG. 17 is finally showna silicon photodiode 508 of the same type as the photodiode 507. Thepurpose of this photodiode is to determine where in its modulation cyclethe radiation source is at a certain moment.

By excitation of a luminophor with a sinus modulated excitationradiation applies for a luminophor undergoing monoexponental decay:

    ω·τ=tan φ

where ω is the angular frequency of the excitation radiation, τ is thelife time of the emission radiation, and φ is the phase shift of theemission radiation. If the phase shift between the emission radiationand the excitation radiation is electronically maintained at 45° (tanφ=1) applies:

    ω=1/τ

and thus, according to the wellknown Stern-Volmer equation:

    ω/ω.sub.0 =1+K.sub.sv ·[O.sub.2 ]

where ω₀ is the angular frequency of the excitation radiation at anoxygen concentration of 0, K_(sv) is the so-called Stern-Volmer constantand [O₂ ] is the oxygen concentration.

For known values of ω₀ and K_(sv) the oxygen content [O₂ ] can thus bedetermined on the basis of detecting the angular frequency ω or thefrequency f=ω/2π of the excitation radiation. In practice the decay isnot mono-exponential and a linear relation between ω (or f) and O₂ istherefore not seen. A reproducible relation has, however, appearedobtainable not just for sinus modulated excitation radiation, but alsofor excitation radiation modulated in other ways, i.e. square wavemodulated excitation radiation.

A simple electronic circuit whereby ω or f can be determined is shown inFIG. 19.

FIG. 18 shows a prototype for an optical unit 60 for use indetermination of the hemoglobin content of a blood sample. The bloodsample is located in the measuring chamber 4007 in the sample container4000.

The sample container 4000 is placed in a holder situated in a section,generally designated 3. Broad-banded radiation is transmitted from aradiation source 601 in the form of a halogen lamp with built-in lens ofthe type LNS-SE6-560 from Hamai Electric Lamp Co. TTD., Tokyo, Japan, toa heat absorbing filter 602 of the type KG5 from Scott. The filter 602eliminates radiation from the infrared range. The radiation istransmitted from there to the measuring chamber 4007 and further to aconvex lens 604, which focus the radiation onto two radiation detectors607 and 609. From the convex lens 604 the radiation is transmitted to adichroic mirror 605 from Optisk Laboratorium, Technical University ofDenmark, Lyngby, Denmark, which reflects radiation of wavelengths lessthan 560 nm and transmits radiation of wavelength greater than 560 nm.

The reflected radiation is transmitted from the dichroic mirror to aband-pass filter 606 (centre value 506 nm; half band width 6 nm;Ferroperm, Vedbaek, Denmark) and from the filter to a radiation detector607 of the type SFH 212 from Siemens. The radiation passing through thedichroic mirror is transmitted to a band-pass filter 608 (centre value600 nm; half band width 6 nm; Ferroperm, Vedbaek, Denmark) and to asilicon photodiode 609 of the type SFH 212 corresponding to the siliconphotodiode 607. Finally a silicon photodiode 603 of the same type as thephotodiodes 607 and 609 receives radiation reflected from differentsurfaces.

The intensity of the radiation source 601 is regulated on the basis ofthe radiation received by the silicon photodiode 603, aiming at aconstant radiation intensity from the radiation source 601. Calculationof the total hemoglobin content Hb_(tot) and the oxygen saturation cansubsequently be fulfilled on the basis of the diode signals. Thecalculation is performed in a wellknown way according to Lambert Beer'sLaw from predetermined values of the extinction coefficients for Hb andHbO₂ or quantities proportional hereto at the relevant wavelengths. Thisdetermination principle is well known from the oxygen saturation meterOSM2 produced and sold by Radiometer A/S, Copenhagen, Denmark.

FIG. 19 shows the electronic circuit coupled to the optical unit shownin FIG. 17, which circuit in a simple way makes it possible to determinethe modulation frequency providing a phase shift of 45° betweenexcitation radiation and emission radiation.

A photodiode 501 is supplied from a voltage controlled oscillator 513with a time modulated voltage signal. The photodiode 501 consequentlyemits time modulated radiation, which excites the luminophor in theluminophor containing PVC coating 5004. Subsequently the luminophoremits time modulated emission radiation, which is phase shifted inrelation to the excitation radiation. The emission radiation falls ontothe photodiode 507 emitting a time modulated current signal which isamplified with a constant quantity in an amplifier 509. The amplifiedsignal is further amplified to a specified amplitude in an amplitudecontrolling unit 510. In a phase detector 511 the phase of signal fromthe unit 510 is compared to the phase of a reference signal, which isphase shifted 45° in relation to the time modulated voltage signalsupplying the photodiode 501. The reference signal is generated in aunit 512. The phase detector 511 emits a signal controlling the voltagecontrolled oscillator 513. By means of the control signal the voltagecontrolled oscillator 513 is adjusted to such a frequency, that theemission radiation--and with this the input signal to the phase detector511 from the unit 510--and the reference signal has the same phase. Inother words, the voltage controlled oscillator 513 is adjusted to such afrequency that the emission radiation is phase shifted 45° in relationto the excitation radiation.

The frequency of the output signal from the voltage controlledoscillator 513 is registered and transformed to a digital quantity inthe unit 516 and/or transformed in a frequency/analogue converter 514 toa signal registered by a printer or plotter 515.

In FIG. 19 the photometric system is for clearness represented only bythe photodiodes 501 and 507. The other not shown components appear inFIG. 17.

With respect to the dimensions of the sample container used in thesystem according to the invention it is noted that each measuringchamber preferably has a volume of 1-50 μl, more preferably 5-25 μl andin particular 7-12 μl and that the distance between the opposedmeasuring chamber walls in the direction of the transmitted radiation ispreferably 5-600 μm, more preferably 8-300 μm and in particular 10-200μm.

Finally, it is noted that the radiation source and the radiationdetector may comprise one component or several components. In the lattercase each component preferably emits respectively detects radiation at adifferent wavelength than the other components. The several componentsof the radiation source may be provided as one integrated device or asseparate devices. The same applies to the radiation detector.

We claim:
 1. A method of photometric in vitro determination of at leastone blood gas parameter in a sample of whole blood obtained directlyfrom an in vivo locality comprising the steps of:(a) connecting an atleast partially transparent sample container to the in vivo locality toobtain a sample of whole blood, the sample container being open at oneend to receive the sample of whole blood and being essentially sealed tothe passage of liquids but permitting the venting of gas at the oppositeend; (b) transferring a sample of whole blood directly from the in vivolocality to the sample container; (c) breaking the connection betweenthe in vivo locality and the sample container; (d) arranging the samplecontainer relative to an optical system, wherein the optical systemcomprises a radiation source and a means for detecting radiation, suchthat the sample container is located between the radiation source andthe radiation detection means; (e) transmitting radiation from theradiation source to the sample; (f) transmitting radiation emitted fromthe sample to the radiation detection means; (g) detecting the radiationtransmitted in step (f); and (h) determining the blood gas parameter ofthe sample of whole blood from the radiation detected in step (g). 2.The method of claim 1, wherein the sample container is placed in asample container station and the sample container station is arrangedrelative to the optical system, such that the sample container islocated between the radiation source and the radiation detection meansof the optical system.